James A. Poupard and Jeff Karr
James A. Poupard is Committee Chair and Jeff Karr is the ASM Archivist at the Center for the History of Microbiology/ ASM Archives, Albin O. Kuhn Library & Gallery, University of Maryland Baltimore County, Baltimore.
There is probably no more universal technique in microbiology than the simple Gram stain. Early American textbooks and manuals describing bacteriological methods that were published between 1880 and 1915 consistently specified that bacteria be stained on cover slips. Many of these books provide great detail on how to prepare cover slips for staining purposes, including the use of special forceps to make the process less cumbersome. However, none explained why stains should be applied to specimens on cover slips rather than on microscope slides. Instead, the procedures described staining then heat fixing specimens on cover slips before placing each cover slip, bacteria side down, on a slide containing a drop of water and reading it with a high-power immersion lens.
One popular early text was The Principles of Bacteriology: A Practical Manual for Students and Physicians by Alexander Abbott, first published in 1892, with the final edition published in 1915. In all nine editions Abbott laid out a standard procedure for staining bacteria that depended on the use of cover slips. In his text, he dedicates more than five pages to describing how to prepare those coverslips, perform the stain, and read the results. Although Abbott provides more detail than can be found in other contemporary texts on this subject, the descriptions in books published in America during the 1880s and 1890s follow this same cumbersome methodology.
Insights into Coverslip Practices
One author from that period who does provide insight as to why specimens were stained on cover slips rather than slides was George M. Sternberg, who is often cited as the first American author to produce a classic, widely accepted textbook on bacteriology for the increasing number of Americans interested in that subject. But first, let us furnish some context and background for this book. In 1880, Sternberg published a translation of Les Bacteries by Antoine Magnin, a book that made no mention of staining techniques. However, Sternberg greatly expanded the 1884 edition of the book, contributing original material that was nearly twice the length of Magnin's original text. Part of Sternberg's new material included advice about producing photomicrographs and also methods for staining bacteria, employing the coverslip method.
Sternberg's major work in this arena, A Manual of Bacteriology, appeared in 1892. That most impressive and comprehensive publication of its kind by an American during the 1890s also addresses the cover-slip issue in some detail:
In Germany it is the custom, in making smear preparations, to press the material between two glass covers, which are then separated by sliding them apart, thus leaving a thin layer upon each. Most bacteriologists make their preparations upon cover glass, as above described, but the writer has for a number of years made his mounts of bacteria upon the glass slide, and believes that this method has some advantages for every-day work. The thin glass covers required when a preparation is to be examined with an immersion objective of high power, are easily broken and often dropped from the fingers or forceps. When the material to be examined is spread and dried directly upon the glass slide, the operation is attended with less difficulty and fewer accidents and the results are quite as good. In this case the slide is held in the fingers during the various steps in the operation of distributing, drying, and staining, while the thin glass cover must be held in delicate forceps.
This textbook, along with Abbott's somewhat more modest work, marks the beginning of American textbook publication in bacteriology. This passage and others like it in the Sternberg book are emblematic of the emergence of American bacteriology from the dominance of the French and German schools. Previously, American scientists relied mainly on visits to Europe to gain access to emerging knowledge about bacteriology, or they used translated texts while paying deference to established European techniques.
Yet here is Sternberg in the Translator's Preface to the 1880 Magnin text regarding the confusing and sometimes contradictory results reported in the French and German bacteriological literature of that time:
This being the condition of affairs, it seems to me that it is necessary for us to commence investigating for ourselves-first making ourselves familiar with what has been done abroad, and then avoiding, if possible, the quicksands into which unfortunate science has too often been dragged by her votaries.
But we should not make too much of Sternberg's call for independence. Despite his rejection of the German staining technique in 1892, almost no other author of an American book published a recommendation to perform the stain on slides. This held true for many years and is reflected in the Alexander Abbott publication (1915), as well as popular books by other bacteriologists, including Edwin O. Jordan (1908) and H. W. Conn (1907). Although there are a few exceptions, like Paul Heinemann (1915) who describes the use of slides as an alternative to cover slip staining, it seems that the practice remained common at least into the 1920s.
Forceps Designed Specifically for Holding Cover Slips
Another indication of the common practice of staining bacterial specimens on coverslips during this period was the development of forceps specifically designed to hold coverslips for staining. One of the more popular designs for this purpose, the Stewart coverslip forceps, was described in 1895 by Alonzo H. Stewart, a demonstrator of clinical microscopy at Jefferson Medical College in Philadelphia. Joseph McFarland described the significance of this innovation when he wrote: "Only those who have experienced the facility with which capillary attraction can draw the staining between the blades of the ordinary forceps from the cover-glass upon which it has been dropped to the fingers, can appreciate this boon."
Stewart graduated from Jefferson Medical College in 1892, and served as demonstrator of clinical microscopy there from 1894 to 1896. He also helped to establish the bacteriology laboratory at Jefferson. Later, he became a bacteriologist at the Bureau of Health of Philadelphia and was an adjunct professor of Bacteriology at the Philadelphia Polyclinic and College for Graduate Medicine.
His innovation was described in a brief article that appeared in 1895 in Medical News. The headline and article are reprinted below:
A NEW COVER-SLIP FORCEPS by A. H. STEWART, M.D., OF PHILADELPHIA; INSTRUCTOR IN CLINICAL MICROSCOPY, JEFFERSON MEDICAL COLLEGE
As the microscope comes into more general use by the physician, and as the study of bacteriology has become so necessary, new devices are constantly being brought forth to aid in research. Many new instruments are needed; many old ones have become useless.
There is no one instrument that is used by bacteriologists so frequently as cover-slip forceps; but every microscopist knows how inefficient are the cheap varieties and how comparatively expensive are the good ones, and none is entirely satisfactory.
The forceps here illustrated has proved in my work more generally satisfactory than any other yet seen. It is made of coppered steel wire or German silver wire, and can be manufactured for about one-fifth the cost of the original Lindslay or the Cornet nickel-plated steel forceps, and is just as steady and durable.
The spring is sufficiently strong to hold firmly a slide or cover- glass without allowing it to move from side to side and yet not powerful enough to break the glass. The blades of the forceps are locked, thus giving a perfectly steady movement and constant apposition when not in use. The extremities of the blades are small and circular and are made in such a way that the cover-slip held in position has a slight inclination downward away from the instrument, so that the stain when placed upon the cover-glass does not run down on the lower blade of the forceps, even when the blades are moist.
When placed upon the table there is no tendency for the instrument to upset, the base being more than an inch wide, and when placed upon the slide the coverglass escapes injury, not striking the table because that end of the forceps is overbalanced by the other.
The comparative cheapness and durability of this forceps will recommend it as a most valuable instrument, not alone for ordinary private work, but in large laboratories where many men are at work, and large numbers of such instruments are required.
It is manufactured by Bausch & Lomb, of Rochester, N.Y., and can be procured from dealers in microscopic supplies in Philadelphia.
It is interesting how a technique that seems so cumbersome by current standards survived for so many years, especially in the face of a strong endorsement from Sternberg in 1892 for the alternative use of slides. This resistance to change demonstrates how techniques employed in Europe greatly influenced early American bacteriologists, who were reluctant to ignore or diverge from following the recommendations from European experts in the new field of bacteriology. We hope that this brief discussion also demonstrates how information found in the Center For the History of Microbiology's Bibliography of Early American Publications in Bacteriology may stimulate others who are interested in the history of bacteriology to become familiar with these significant early publications.
Abbott, A. C. 1892. The principles of bacteriology: a practical manual for students and physicians. Philadelphia, Lea Brothers and Co.
Abbott, A. C. 1915. The principles of bacteriology: a practical manual for students and physicians. 9th ed., Philadelphia, Lea Brothers and Co.
Conn, H.W. 1907. Practical dairy bacteriology, prepared for the use of students, dairymen, and all interested in the problems of the relation of milk to public health. New York, Orange Judd Co.
Jordan, E. O. 1908. A text-book of general bacteriology. Philadelphia and London, W. B. Saunders Co.
Heinemann, P. G. 1915. A laboratory guide to bacteriology, 3rd ed. University of Chicago Press, Chicago.
Karr, J., and J. Poupard. 2010. The center for the history of microbiology bibliography of microbiology-related publications in America: 1721 to 1915. http://www.asm.org//images/Membership/archives/bib%20project.pdf Center for the History of Microbiology/ASM Archives (CHOMA) Bibliography of Early Microbiology Publications
Magnin, A. 1880. The bacteria. Translated by George M. Sternberg. Boston, Little, Brown, and Co.
Magnin, A., and G. M. Sternberg. 1884. Bacteria. New York, W. Wood and Co.
McFarland, J. 1937. The beginning of bacteriology in Philadelphia. Bull. Hist. Med. 5:149-198.
Poupard, J. A. 2010. A history of microbiology in Philadelphia. Bloomington, IN Xlibris. p. 55.
Sternberg, G. M. 1892. A manual of bacteriology. New York, W. Wood and Co.